1. Field of the Invention
The present invention relates to the field of fluidic devices for carrying out multiplex chemical or biochemical reactions and for performing multiplex chemical and/or biochemical assays. More particularly, this invention relates to devices and methods for distributing fluids into a plurality of compartments for carrying out multiplex chemical and/or biochemical reactions, and detecting a plurality of chemical and/or biochemical compounds.
2. Description of Related Art
Modern drug development, disease diagnosis, pathogen detection, gene discovery, and various genetic-related technologies and research increasingly rely on making, screening, and assaying a large number of chemical and/or biochemical compounds. Traditional methods of making and examining the compounds one at a time are becoming increasingly inadequate. Therefore there is a need for chemical/biochemical reaction systems and devices to perform high-throughput assay and synthesis.
One of the most commonly used high-throughput multiplexing method relies on the use of titer plates. Each titer plate contains 96, 384, or 1,536 microwells or microtubes in which individual chemical and/or biochemical reactions are carried out. (need a reference) In a standard format the reaction media inside individual microwells or microtubes are physically isolated from each other. Chemical and biochemical reagents are delivered into the microwells or microtubes either robotically or manually using pipettes or dispensers. In a standard format the distances between adjacent microwells or microtubes are 9.0 mm, 4.5 mm, and 2.25 mm for 96, 384, and 1,536 microwell titer plates, respectively. To increase throughput, higher densities of the microwells are needed.
Another multiplexing method relates to microarrays. The most well-known microarray is DNA microarray, which, in its most common form, is a glass plate containing a two-dimensional array of DNA materials on its surface. A DNA microarray is used as a multiplexing detection device. Each element of the array has a unique DNA sequence, which is used to specifically recognize or detect a unique complementary DNA sequence in a sample solution. The element density of a DNA microarray is usually much higher than that of a titer plate. On a commercially available DNA microarray the distance between two adjacent elements is between 10 micrometer and 500 micrometer. DNA microarray, are rapidly becoming fundamental tools in genomic, proteomic, and other biological research (Fodor et al. Science 251, 767 (1991), Schena et al. Science 270, 467 (1995) and “The Chipping Forecast II” Nat. Genet. 32 (2002)). In addition to research use, DNA microarray has the potential to be used as a clinical diagnostic tool (Carr et al. Nat. Oncogene. 22, 3076 (2003) and “Microarrays in Cancer: Research and Applications” BioTechniques Supplement March 2003). In addition to DNA microarray there are various other types of microarrays, such as peptide microarray, protein microarray, and tissue microarray, for various research and diagnostic applications (Gao et al. Nature Biotechnol. 20, 922 (2002)).
Microarray technology has fundamentally changed the way of studying biological systems from observing one or a few genes or molecular species at a time to observing pathways, networks, and molecular machines that involve the interplay of a large collection of genes and pools of molecules. DNA microarray chips available today operate based on the hybridization of target DNA or RNA molecules (the sample to be tested) in a solution phase with probe DNA (oligonucleotides or cDNA) molecules immobilized on solid substrates, which are mostly in either plate or bead forms (Rubenstein in BioTechniques Supplement March 2003). The hybridization results are used in monitoring gene expression, determining nucleotide sequences, identifying gene mutations, detecting pathogens, and selecting and measuring activities of ligand molecules such as peptides, proteins, antibiotics and other organic and inorganic molecules.
In spite of the usefulness of the currently available DNA microarrays, their performance is far from being satisfactory for many applications. Inadequate assay specificity is one of a multitude of limitations with the current DNA microarray methodology, which are fundamentally associated with the single-pair hybridization assay, i.e. with results determined by the hybridization of only one pair of nucleotide molecules. Assay specificity relies on hybridization discrimination, which in turn is determined by probe (immobilized DNA) sequence design, probe sequence purity, target (sample DNA) sequence composition, and hybridization conditions. Selection of hybridization probes is a complex issue, particularly for gene expression applications, in which samples contain tens of thousands genes. Shorter oligo probes should theoretically provide higher hybridization discrimination but they tend to have poor hybridization properties leading to lower sensitivity, not to mention the difficulty of finding short unique sequences in large genomes (Shchepinov et al. Nucleic Acids Res. 25, 1155 (1997) and Hughes et al. Nat. Biotechnology, 19, 342 (2001)). As oligo probes become longer, the hybridization discrimination decreases, although detection sensitivity increases and it is easier to find unique sequences in large genomes. It has been found that when the probe length reaches 35, it needs to have at least 3 mismatches to reliably discriminate different target DNA sequences by hybridization. This fundamental problem of limited specificity has lead to different results from chips of different venders and technology platforms (Kuo et al. Bioinformatics 18, 405 (2002)).
Today's DNA microarrays are not suitable for quantitative measurement. This will likely become one of the roadblocks to hinder the technology from being used as a clinical diagnostic tool, although technological efforts have been made to address this problem (Dudley et al. Proc. Natl. Acad. Sci. 99, 7554 (2002)). Studies have shown a significant compression of differential ratios (ratios of hybridization intensities from different samples) in microarray data as compared to real-time PCR (Polymerase Chain Reaction) data. Real-time PCR has been established as the most commonly used and accepted standard for validating DNA microarrays in gene expression use (Chuaqui et al. Nat. Genet. 32 Supplement 509 (2002)). According to the published data, while about 70% of array results of highly differentiated genes were qualitatively consistent with real-time PCR, consistent validation was not achieved for genes showing less than a four-fold change on the array. For many of the genes examined, significant quantitative differences were found between array- and real-time-PCR-based data (Mangalathu et al. Journal of Molecular Diagnostics 3, 26 (2001)). For these reasons, array users often choose for further study only those genes with the highest differential expression ratios. This strategy can easily overlook genes of significant interest. Obviously, it is highly desirable to develop a more robust and quantitative array platform in order to reach a level of confidence for which relatively small differences in gene expression between samples are real and that genes showing such differences are worth further investigation.
The third limitation of today's DNA microarray is detection sensitivity. The single-pair hybridization assay used in the DNA microarray does not involve any amplification and requires a fairly large amount of sample. For example, in gene expression applications with most of the commercial array products, 2 to 5 microgram of total RNA sample is needed for each assay. However, some of the clinical biopsy tissue samples yield less than 1 microgram of total RNA sample. For pathogen detection, microarrays are considered not sensitive enough without the aid of PCR (Call et al. J Microbiol Methods 53, 235 (2003)). Amplification of either DNA or RNA samples during sample preparation has been used to boost the amount of samples before they are applied to array chips (Lockhart et al. Nature Biotech. 14, 1675 (1996)). This method, however, causes concerns for altering ratios of the genes involved.
The challenges of specificity, accuracy, and sensitivity mentioned above can be solved using real-time PCR. Higuchi et al. first demonstrated fluorescence monitoring kinetic PCR amplification process in real-time (Higuchi et al. Biotechnology 10, 413 (1992)). The method has been developed into a powerful tool, often referred as a golden standard, for quantitative measurement of nucleic acids with various applications, including gene expression, pathogen detection, and SNP (Single Nucleotide Polymorphism) detection. Due to its reduced detection time and simplification of quantification, the method is believed to potentially have the greatest impact on the general public in environmental monitoring and nucleic acid diagnostics (Walker, Science 296, 557 (2002)).
A real-time PCR system detects PCR products as they accumulate during a PCR reaction process. There are several variations of detection systems. The most well-known and popular system is Taqman system (Heid et al. Genome Res. 6, 986 (1996)). A pair of PCR primers and one fluorescence resonance energy transfer (FRET) probe are used in the detection of each target sequence. The FRET probe is a short oligonucleotide complementary to one of the strands of the target sequence. Each FRET probe contains a reporter dye and a quencher dye. Taq polymerase is used. If the target sequence is present, the probe anneals downstream from the forward primer site and is cleaved by the 5′ nuclease activity of Taq DNA polymerase as this primer is extended. The cleavage of the probe separates the reporter dye from quencher dye, increasing the reporter dye signal and allowing primer extension to continue to the end of the template strand. Additional reporter dye molecules are cleaved from their respective probes with each cycle, causing an increase in fluorescence intensity proportional to the amount of amplicon produced.
Real-time PCR assay is intrinsically highly specific. For one target sequence to be detected, it has to contain all three sequence segments complementary to a detection probe, a forward primer, and a reverse primer, respectively. Any errors produced by one event will likely be filtered out by the other two events. For example, if in one event a forward primer happened to prime to a wrong sample sequence and produced a wrong amplicon, this wrong amplicon will likely either not be recognized by the detection probe or not be further amplified by the reverse primer. In comparison, today's DNA microarrays rely on the hybridization of only one pair of nucleotides and do not have any build-in error-checking mechanism. Even with the multiple-probe approach, such as the one used by Affymetrix (www.affymetrix.com), the assay specificity is not increased in any way and the improvement is only in the reduction of the statistical variance of the data. The benefit of this approach is derived by averaging the results of hybridization of multiple individual probes, which hybridize directly with sample sequences and have no relationship with the hybridization events of any other probes that are designed to target at the same sample sequence or same gene.
Real-time PCR assay is highly sensitive and is quantitative. PCR is an exponential amplification process. In principle, PCR can pick up and amplify a single copy of a target sequence. As a daily practice for RNA detection, real-time PCR requires nanograms of RNA samples as compared to micrograms required by today's DNA microarrays. Moreover, the ability of real-time PCR to quantitatively measure the copy numbers of target sequences in samples is non-existent in today's DNA microarray technology.
Most of existing instruments perform PCR reactions in either 96- or 384-well titer plates. Samples are manually or robotically pipetted into individual wells. Applied Biosystems recently started the sale of a Micro Fluidic Card in a 384-well format (www.appliedbiosystems.com). The new card offers the advantages of reduced consumption of samples and reagents and the elimination of labor-intensive pipetting steps. The new card has the same area size as that of conventional 96- and 384-well titer plates. However, its fluidic design and the operational principle fundamentally limit it from being able to achieve the degree of miniaturization and the level of area density that have demonstrated in DNA microarrays (U.S. Pat. No. 6,272,939).
There have been an increasing number of reports of the development of micro-fabricated PCR devices, including continuous flow and microwell devices made from silicon or plastic materials (Kopp et al. Science 280, 1046 (1998), Nagai et al. Anal. Chem. 73, 1043 (2001), and Yang et al. Lab on a Chip, 2, 179 (2002)). A low-energy consumption and fast thermal cycling silicon-chip-based real-time PCR detection system for field use was also demonstrated (Belgrader et al. Science, 284, 449 (1999)). There are also reports of performing DNA microarray assays using PCR as a sample preparation process involving microfabricated array chips (U.S. Pat. No. 6,448,064). O'Keefe et al disclosed a method for conducting multiple simultaneous micro-volume chemical and biochemical reactions on an array of micro-holes as described in United States Patent Application Publication 2001/0055765 A1. The method is said to be able to perform real-time PCR among several other applications.
For research and many other applications, it is highly desirable to have a flexible way of multiplex synthesis of microarrays of various molecules, including nucleic acids and peptides, and to perform assays of various sequences in a short turn-around time. Gao et al. in U.S. Pat. No. 6,426,184 described a method of combining PGR (photogenerated reagent) chemistry, micromirror array projector, and microwell plates to achieve flexible and highly parallel synthesis of microarrays of varieties of molecules. The teaching of which is incorporated herein by reference. In a separate disclosure, PCT WO 0202227, Zhou described a microfluidic device that has the features of dynamic isolation for performing parallel chemical synthesis using PGR chemistry with improved process robustness. The teaching of the disclosure is also incorporated herein by reference. For the purpose of performing real-time PCR and certain other biochemical assays in a microarray format and in a highly multiplexing scale, it is desirable or even necessary to have a build-in static isolation mechanism in a microarray device in addition to a flexible chemical synthesis capability for implementing biochemical probes.
An objective of this invention is to provide microfluidic devices for performing multiplex chemical and biochemical reactions. Another objective of this invention is to provide highly flexible method of implanting a plurality of chemical and/or biochemical molecules into the microfluidic devices. Yet another objective of this invention is to provide methods of multiplex biochemical assays using the microfluidic devices. A further objective of this invention is to provide systems for performing parallel chemical and biochemical assay analysis, including real-time PCR, ELISA (enzyme linked-immunosorbent assay) and other assays.